scholarly journals Degraded Collagen Fragments Promote Rapid Disassembly of Smooth Muscle Focal Adhesions That Correlates with Cleavage of Pp125FAK, Paxillin, and Talin

1999 ◽  
Vol 147 (3) ◽  
pp. 619-630 ◽  
Author(s):  
Neil O. Carragher ◽  
Bodo Levkau ◽  
Russell Ross ◽  
Elaine W. Raines
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesion ◽  
Type I Collagen ◽  
Focal Adhesions ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Type I ◽  
Focal Adhesion Complex ◽  
Adhesion Complex

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.

Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Martin Liu ◽  
Angelos Karagiannis ◽  
Matthew Sis ◽  
Srivatsan Kidambi ◽  
Yiannis Chatzizisis
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Gels ◽  
Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

scholarly journals Restructuring of Focal Adhesion Plaques by Pi 3-Kinase

2000 ◽  
Vol 150 (3) ◽  
pp. 627-642 ◽  
Author(s):  
Jeffrey A. Greenwood ◽  
Anne B. Theibert ◽  
Glenn D. Prestwich ◽  
Joanne E. Murphy-Ullrich
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Adhesive Strength ◽  
Focal Adhesions ◽  
Lipid Product ◽  
Phosphoinositide 3 Kinase ◽  
Triton X ◽  
Adhesion Plaque ◽  
Adhesion Complex

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of α-actinin and vinculin from the focal adhesion complex to the Triton X-100–soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P3 binds to α-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of α-actinin with the integrin β subunit, and that PtdIns (3,4,5)-P3 could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P3, has important implications for the regulation of cellular adhesive strength and motility.

scholarly journals Basic fibroblast growth factor modulates proliferation and collagen expression in urinary bladder smooth muscle cells

2007 ◽  
Vol 293 (4) ◽  
pp. F1007-F1017 ◽  
Author(s):  
Masaaki Imamura ◽  
Akihiro Kanematsu ◽  
Shingo Yamamoto ◽  
Yu Kimura ◽  
Isao Kanatani ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Type I Collagen ◽  
Type Iii Collagen ◽  
Type I ◽  
Fibroblast Growth ◽  
Bladder Smooth Muscle ◽  
Type Iii ◽  
Collagen Expression

Bladder hypertrophy is a general consequence of bladder outlet obstruction (BOO) and a typical phenomenon observed in clinical urologic diseases such as benign prostatic hyperplasia and neurogenic bladder. It is characterized by smooth muscle hyperplasia, altered extracellular matrix composition, and increased contractile function. Various growth factors are likely involved in hypertrophic pathophysiology, but their functions remain unknown. In this report, the role of basic fibroblast growth factor (bFGF) was investigated using a rat bladder smooth muscle cell (BSMC) culture system and an original animal model, in which bFGF was released from a gelatin hydrogel directly onto rat bladders. bFGF treatment promoted BSMC proliferation both in vitro and in vivo. In vitro, bFGF downregulated the expression of type I collagen, but upregulated type III collagen. ERK1/2, but not p38MAPK, was activated by bFGF, whereas inhibition of ERK1/2 by PD98059 reversed bFGF-induced BSMC proliferation, type I collagen downregulation, and type III collagen upregulation. In the in vivo release model, bFGF upregulated type III collagen and increased the contractile force of treated bladders. In parallel with these findings, hypertrophied rat bladders created by urethral constriction showed increased urothelial bFGF expression, BSMC proliferation, and increased type III collagen expression compared with sham-operated rats. These data suggest that bFGF from the urothelium could act as a paracrine signal that stimulates the proliferation and matrix production of BSMC, thereby contributing to the hypertrophic remodeling of the smooth muscle layer.

scholarly journals Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component.

1994 ◽  
Vol 5 (2) ◽  
pp. 183-192 ◽  
Author(s):  
A Woods ◽  
J R Couchman
Keyword(s):  
Heparan Sulfate ◽  
Focal Adhesion ◽  
Type I Collagen ◽  
Core Protein ◽  
Mammalian Species ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Type I ◽  
Adhesion Component

Focal adhesion formation in fibroblasts results from complex transmembrane signaling processes initiated by extracellular matrix molecules. Although a role for integrins with attendant tyrosine kinases has been established, there is evidence that cell surface heparan sulfate proteoglycans (HSPGs) are also involved with an associated role of protein kinase C. The identity of the proteoglycan has remained elusive, but we now report that syndecan 4 (ryudocan/amphiglycan) is present in focal adhesions of a number of cell types. Affinity-purified antibodies raised against a unique portion of the cytoplasmic domain of syndecan 4 core protein recognized an HSPG of similar characteristics to those of syndecan 4. These antibodies stained focal adhesions only after cell permeabilization and recognized differing mammalian species. Syndecan 4 was associated with focal adhesions that contained either beta 1 or beta 3 integrin subunits and those that formed on substrates of fibronectin, laminin, vitronectin, or type I collagen. No focal adhesions were found that were vinculin-containing but lacked syndecan 4. In contrast, syndecan 2, whose cytoplasmic domain is closely homologous to syndecan 4, does not appear to be a focal adhesion component. Thus, syndecan 4 represents a new transmembrane focal adhesion component, probably involved in their assembly.

scholarly journals Overexpression of Decorin by Rat Arterial Smooth Muscle Cells Enhances Contraction of Type I Collagen In Vitro

2004 ◽  
Vol 24 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Hannu Järveläinen ◽  
Robert B. Vernon ◽  
Michel D. Gooden ◽  
Aleksandar Francki ◽  
Stephanie Lara ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Muscle Cells ◽  
Type I ◽  
Arterial Smooth Muscle ◽  
Arterial Smooth Muscle Cells

scholarly journals Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

2008 ◽  
Vol 295 (1) ◽  
pp. C268-C278 ◽  
Author(s):  
Zhe Sun ◽  
Luis A. Martinez-Lemus ◽  
Michael A. Hill ◽  
Gerald A. Meininger
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Mechanical Activity ◽  
Tissue Blood Flow ◽  
Cell Contact ◽  
Type I ◽  
Adhesion Sites

Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2∼5 μm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of α5- and β3-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward ( z-axis) pulling force (800∼1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to α5β1- and αvβ3-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces.

scholarly journals The Mechanosensing and Global DNA Methylation of Human Osteoblasts on MEW Fibers

Nanomaterials ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2943
Author(s):  
Pingping Han ◽  
Cedryck Vaquette ◽  
Abdalla Abdal-hay ◽  
Sašo Ivanovski
Keyword(s):  
Dna Methylation ◽  
Osteogenic Differentiation ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Global Dna Methylation ◽  
Focal Adhesion Complex ◽  
Osteogenic Induction ◽  
Sense And Respond ◽  
Adhesion Complex

Cells interact with 3D fibrous platform topography via a nano-scaled focal adhesion complex, and more research is required on how osteoblasts sense and respond to random and aligned fibers through nano-sized focal adhesions and their downstream events. The present study assessed human primary osteoblast cells’ sensing and response to random and aligned medical-grade polycaprolactone (PCL) fibrous 3D scaffolds fabricated via the melt electrowriting (MEW) technique. Cells cultured on a tissue culture plate (TCP) were used as 2D controls. Compared to 2D TCP, 3D MEW fibrous substrates led to immature vinculin focal adhesion formation and significantly reduced nuclear localization of the mechanosensor-yes-associated protein (YAP). Notably, aligned MEW fibers induced elongated cell and nucleus shape and highly activated global DNA methylation of 5-methylcytosine, 5-hydroxymethylcytosine, and N-6 methylated deoxyadenosine compared to the random fibers. Furthermore, although osteogenic markers (osterix-OSX and bone sialoprotein-BSP) were significantly enhanced in PCL-R and PCL-A groups at seven days post-osteogenic differentiation, calcium deposits on all seeded samples did not show a difference after normalizing for DNA content after three weeks of osteogenic induction. Overall, our study linked 3D extracellular fiber alignment to nano-focal adhesion complex, nuclear mechanosensing, DNA epigenetics at an early point (24 h), and longer-term changes in osteoblast osteogenic differentiation.

scholarly journals FAK Structure and Regulation by Membrane Interactions and Force in Focal Adhesions

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 179 ◽  
Author(s):  
Paula Tapial Martínez ◽  
Pilar López Navajas ◽  
Daniel Lietha
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Structural Changes ◽  
Membrane Lipids ◽  
Current Knowledge ◽  
Focal Adhesions ◽  
Structural Features ◽  
Focal Adhesion Complex ◽  
Patient Prognosis ◽  
Adhesion Complex

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase with key roles in the regulation of cell adhesion migration, proliferation and survival. In cancer FAK is a major driver of invasion and metastasis and its upregulation is associated with poor patient prognosis. FAK is autoinhibited in the cytosol, but activated upon localisation into a protein complex, known as focal adhesion complex. This complex forms upon cell adhesion to the extracellular matrix (ECM) at the cytoplasmic side of the plasma membrane at sites of ECM attachment. FAK is anchored to the complex via multiple sites, including direct interactions with specific membrane lipids and connector proteins that attach focal adhesions to the actin cytoskeleton. In migrating cells, the contraction of actomyosin stress fibres attached to the focal adhesion complex apply a force to the complex, which is likely transmitted to the FAK protein, causing stretching of the FAK molecule. In this review we discuss the current knowledge of the FAK structure and how specific structural features are involved in the regulation of FAK signalling. We focus on two major regulatory mechanisms known to contribute to FAK activation, namely interactions with membrane lipids and stretching forces applied to FAK, and discuss how they might induce structural changes that facilitate FAK activation.

scholarly journals Lipoxin Alleviates Diabetic Vascular Calcification via the YAP Pathway

2021 ◽  
Author(s):  
Baihe Han ◽  
Mengyue Yang ◽  
Qi Liu ◽  
Gang Wang ◽  
Xiaoxue Ma ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Type I Collagen ◽  
Muscle Cells ◽  
Subcellular Localisation ◽  
Type I ◽  
Alizarin Red ◽  
Effective Prevention

Abstract Background: Vascular calcification is highly prevalent in patients with diabetes and has detrimental consequences. However, no effective prevention and treatment methods are currently available. Extensive evidence has demonstrated the protective effect of lipoxin (LX) against vascular diseases. However, whether LX prevents diabetic vascular calcification remains unknown. Here, we tested the hypothesis that LX alleviated osteogenic differentiation and subsequent calcification of vascular smooth muscle cells (VSMCs).Methods: In vitro, human aortic smooth muscle cells (HASMCs) were incubated in osteogenic medium (OM) with advanced glycation end products (AGEs) and LX to further determine the underlying mechanisms. An in vivo diabetic mouse model was established using a combination of a high-fat diet and multiple formulations of low-dose streptozotocin (STZ). Cell culture, alkaline phosphatase (ALP) staining, ALP activity, Alizarin red staining, von kossa staining, determination of calcium content, western blot analysis, immunohistochemistry, and immunofluorescence staining and statistical analysis were used in our study. Results: AGEs dose-dependently induced calcification and expression of osteogenesis-related markers, including Runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and type I collagen (COL1), coupled with the activation of yes-associated protein (YAP). Mechanistically, YAP activation enhanced the AGE-induced osteogenic phenotype and calcification, but inhibition of YAP signalling alleviated this trend. Consistent with the in vitro results, diabetes promoted YAP expression as well as the subcellular localisation of the protein in the nucleus in the arterial tunica media. Interestingly, treatment with LX reduced vascular osteogenesis and calcification in diabetic mice, which was correlated with the reduced YAP levels. In addition, LX significantly inhibited COL1 accumulation and modulated the extracellular matrix. Our results further demonstrated that a pharmacological agonist of YAP reversed LX-mediated protection against osteogenic phenotypic conversion and calcification in VSMCs.Conclusions: These results demonstrate that LX attenuates transdifferentiation and calcification of VSMCs in diabetes mellitus via the YAP signalling axis, suggesting that LX is a potent therapeutic strategy to prevent diabetic vascular calcification.

scholarly journals Mechanotransduction through fibronectin-integrin focal adhesion in microvascular smooth muscle cells: is calcium essential?

2012 ◽  
Vol 302 (10) ◽  
pp. H1965-H1973 ◽  
Author(s):  
Zhe Sun ◽  
Zhaohui Li ◽  
Gerald A. Meininger
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Focal Adhesion ◽  
Kinase Inhibitor ◽  
Contractile Response ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Mechanical Force ◽  
Type I ◽  
Low Passage

It is believed that increased transmural pressure exerts force on vascular smooth muscle cells (VSMCs) and triggers Ca2+ signaling as an initiating event responsible for the arteriolar myogenic response. However, the mechanisms linking the pressure increase to Ca2+ signaling are unclear. We have shown previously using atomic force microscopy (AFM) that mechanical force induces a VSMC contractile response when applied to single fibronectin (FN; Sun Z, Martinez-Lemus LA, Hill MA, Meininger GA. Am J Physiol Cell Physiol 295; C268–C278, 2008) focal adhesion sites. This current study seeks to determine whether application of force to single focal adhesions can cause a change in VSMC Ca2+. Experiments were performed in low passage (p3∼10) as well as in freshly isolated skeletal muscle arteriole VSMCs. AFM-attached microbeads (5 μm) were coated with FN or collagen type I (CN-I) or type IV (CN-IV) and placed on a VSMC for 20 min, resulting in formation of a focal adhesion between the cell and the microbead. In low passage VSMCs, mechanically pulling on the FN-coated beads (800∼3000 pN) did not induce a Ca2+ increase but did cause a contractile response. In freshly isolated VSMCs, application of an FN or CN-I-coated bead onto the cell surface induced global Ca2+ increases. However, these Ca2+ increases were not correlated with the application of AFM pulling force to the bead or with the VSMC contractile responses to FN-coupled pulling. Chelating cytosolic Ca2+ using BAPTA loading had no negative effect on the focal adhesion-related contractile response in both freshly isolated and low passage VSMCs, while the Rho-kinase inhibitor Y27632 abolished the micromyogenic response in both cases. These observations suggest that, in freshly isolated and cultured VSMCs, application of mechanical force to a focal adhesion does not invoke an acute global Ca2+ increase. On the other hand, our data support a role for Rho-linked signaling mechanism involved in mechanotransduction leading to focal contraction that is independent of the need for a global increase in VSMC Ca2+.

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